This application's long- term specific aims are to elucidate mechanisms whereby Staphylococcus epidermidis and other coagulase-negative staphylococci (CoNS) have become one of the most important nosocomial pathogens during the past decade. These investigations focus on 2 areas: 1) microbial virulence factors that allow these-organisms to adhere to, persist on, and disseminate from prosthetic devices and intravascular catheters and 2) host mechanisms of resistance that could be augmented by active or passive immune based treatments. The bacterial capsule polysaccharide/adhesion (PS/A) is an important virulence factor in that it promotes most of the early, initial attachment of CoNS to a variety of biomaterials and is critical for resistance to phagocytosis and blood borne dissemination. PS/A has been characterized at the chemical and immunologic level, and a serologically conserved antigen is commonly expressed by most clinical isolates of CoNS. Isogeneic strains made deficient in PS/A production by transposon (Tn) mutagenesis have been used for comparisons of in vitro and in vivo pathogenic properties. These investigations will be extended to the following areas related to elaboration of PS/A: characterize the DNA involved in production of PS/A by cloning a likely PS/A-gene cluster identified by Tn insertions, as well as fragments of DNA within this cluster flanking the Tn inserts; determine the nucleotide sequence of this genetic region; use genetic complementation to identify genes involved in PS/A production; analyze RNA transcripts to define the organization of the PS/A gene cluster; and evaluate whether the encounter of PS/A-producing S. epidermidis with either biomaterials or skin affects PS/A production by use of transcriptional fusions of beta galactosidase to PS/A-genes. Because PS/A is also the bacterial capsule, it protects these cells from host defenses and is a target for protective antibodies. This application proposes a comprehensive evaluation of the mechanisms whereby PS/A protects bacterial cells from host defenses and how PS/A- specific antibodies augment immunity. Studies proposed include: determining the mechanism whereby PS/A interferes with deposition of C3 opsonins onto the bacterial surface; evaluating how antibodies to PS/A overcome bacterial resistance to immunity; assessing whether PS/A- specific antibodies can opsonize S. epidermidis cells growing in a biofilm for phagocytic killing; studying the ability of PS/A-positive and -negative strains to spread from a contaminated intracardiac foreign body to adjacent aortic valve tissue; and finally, determining whether vaccination with PS/A, or passive infusion of an antibody to PS/A, protects animals against disease when infection occurs in a manner analogous to surgical contamination during implantation of a foreign body. The last aim is to prepare a PS/A vaccine for phase I safety and immunogenicity evaluations in humans. These studies should further the understanding of why S. epidermidis and related CoNS are so commonly associated with device infections, and the potential of immunoprophylaxis for lowering the occurrence of nosocomial coagulase-negative staphylococcal disease.